This invention relates to an apparatus for the culture of cells. The cells to be cultured in this invention are viable, growing or non-growing, prokaryotic and eukaryotic cells such as bacteria, yeast, plant, animal and human cells. These cells may be derived in any manner, that is, isolated from nature, mutated, in the naturally-occurring form, genetically engineered or modified, transformed or non-transformed, hybrids formed by fusion between portions of cells or whole cells of the same or different species. These cells may be attached to the substrate, grown in suspension, or in suspension attached to or within another substrate, such as microcarrier beads or immobilized in some other manner. The cultures may consist of a single cell line or a plurality of cell lines of the same or different species.
Cell lines which produce such proteins as blood factors, interferons, growth hormones and lymphokines are very sensitive to chemical and mechanical stresses (particularly shear forces). Hence their propagation in conventional bioreactors developed for the cultivation of microorganisms with a rigid cell wall is difficult. Many existing bioreactors for animal cell culture have been designed on the principles originally developed for microbial culture, herein referred to as modified microbiol fermentor (MMF) devices (J. Van Brunt, Biotechnology 5: 1134-1138, 1987). These fermentors are aerated by gas overlay and/or sparged air through an open pipe or an open pipe or perforated ring at the bottom of the compartment. Agitation is accomplished by either blade impellers, sail impellers or floating stainless steel mesh stirrers to increase oxygen transfer from gas overlay. These fermentors can also include high-speed rotating stainless steel mesh cylinders for perfusion. These latter means of agitation generally impart turbulent flow characteristics. The bases of the vessels range from flat to slightly rounded to hemispherical. Some of the adaptations of hardware (e.g. hemispherical base) have been successful although these particular animal cell culture devices are limited to only certain types of cells. The major drawback of the MMF is the fluid and mechanical shearing associated with the sparged air and agitation impellers used for gas transfer and minimizing zones of excess nutrients or titrants (pH control).
Other devices utilizing indirect gas transfer, such as gas-permeable membranes or `caged` aeration systems have been developed. The design features including the use of silicone tubing windings (see: U.S. Pat. No. 4,649,114) and stainless steel mesh cylinders (see: U.S. Pat. No. 4,727,040) would be technically difficult or economically prohibitive on scale-up units. The manufacturing costs for large scale fine-mesh components of the gas exchange system could be very high. As well, problems related to shear forces generated due to rotation of the cylinder, which minimizes biofouling, are detrimental to cell integrity (A. J. Brennan, New Brunswick Tech. Bull. D-01406-02-87, 1987). Agitation is provided by a blade impeller or pressure-differential. The bases of the vessels range from flat to slightly rounded to hemispherical.
Classical airlift systems with a concentrically-placed, or occasionally non-concentric configuration, draft tube or component with similar function within the vessel have been implemented for animal cell culture (J. Van Brunt, 1987, op.cit.). These systems tend to induce strong fluid shear forces which can be extremely detrimental to growth and/or productivity. Normally these systems drive air in at the base of the vessel to create a density difference in the liquid. The rising liquid not only imparts oxygen for growth and metabolism but also lifts the cells and mixes the liquid. However, as the bubbles rise they coalesce into larger bubbles and upon contacting the surface of the liquid the bursting bubbles create extreme shear stress on the cells (bubble shear) leading to metabolic stress or even cell destruction.
There has recently been a report of a bubble-free aeration system (R. Wagner and J. Lehmann, TIBTECH 6(5), 101-104, 1988). This system comprises a hydrophobic membrane made of polypropylene that is formed as a porous hollow fiber. Bubble-free aeration is achieved if the internal gas pressure does not exceed the pressure at which the bubbles will form. The hydrophobic membrane is looped around a carrier that is slowly moved through the culture to produce a membrane stirrer. This system would be difficult to scale-up. Cells and microcarriers would probably become trapped on the membrane. Dead zones would be present within the system and the hydrodynamics would be unpredictable.
Another alternative technology for animal cell culture is fluidized bed reactors. The cells are immobilized by hydrogel encapsulation or entrapment and air is sparged at the base of the vessel. The vessels have a large height to diameter ratio and have cylindrical or conical bases. The immobilization requirements limit the system's versatility and would present difficulties in scale-up.
Cells have also been immobilized on an inorganic cylindrical (ceramic) support matrix with micro-channels for direct infusion of oxygenated medium. Such support matrices cannot be reused. Scale-up of such a system would be expensive with labor intensive operation and maintenance.
U.S. Pat. No. 4,661,458 teaches a system wherein the cells are provided on an organic tubular or laminate membrane cell support. Such a system would provide for the formation of non-homogeneous microenvironments. The growth of cells on such a support can impede mass transfer of nutrients and gases.
In summary, problems with currently available cell culture technologies include: fluid and mechanical shear; supply of oxygen; measurement and control of the system; formation of gradients (pH, dissolved oxygen, temperature and nutrients); removal of products and wastes, gaseous or non-gaseous in nature; versatility and potential for scale-up.
The ideal animal cell bioreactor design requires:
(a) An agitation system which provides gentle and predictable flow patterns and optimized mass transfer. Mixing must also be sufficient to minimize gradients within the vessel while avoiding mechanical shear. PA1 (b) The use of indirect gas transfer through a gas-permeable membrane because of the fluid and mechanical shear associated with the aeration systems of existing technologies. PA1 (c) That there be ideally effective real-time measurement and control of growth and production parameters. PA1 (d) A means for product and waste product removal which is not affected over long runs by biofouling of the device. PA1 (e) That the system be versatile; ideally it should be applicable to shear-resistant and shear-sensitive cell lines in microcarrier and suspension culture. The bioreactor should be able to operate in the batch, fed-batch, repeated fed-batch, perfusion and continuous modes. PA1 (f) That the system be scaleable. PA1 (a) a cell culture compartment having a side surface and two opposite flow-directing surfaces defining together a low-turbulence internal compartment surface, PA1 (b) a compensation chamber disposed above said compartment in fluid communication therewith, PA1 (c) a gas exchange tube disposed within said compartment and having opposite open ends facing each one of the flow directing surfaces, the gas exchange tube having an inner surface and an outer surface, both surfaces being provided with gas exchange means for supplying and removing gases to and from the culture medium, PA1 (d) gas conduit means communicating with the gas exchange means from outside the compartment; and PA1 (e) liquid-lifting means disposed within said compartment substantially coaxially with the gas exchange tube.
A scaleable bioreactor incorporating the above-mentioned features having capability to be operated in various modes as outlined would be desirable.